Although V3 sequences of HIV-1 are the principal genetic determinants of the viral cell specificity, little is known about the role of SIV V3 sequences. To understand the role of SIV V3 sequences, we have focused on a molecularly cloned SIV, designated as EvT3, which displays unique V3 sequences, cell specificity in vitro and pathogenic potential in vivo. Compared to SIVmac239, EvT3 V3 has six amino acid substitutions within the 32 amino acid stretch of SIV V3. In culture, EvT3 does not replicate in human T cell lines that are highly sensitive to SIVmac239 infection. When experimentally infected into rhesus macaques, EvT3, but not SIVmac239, induces severe and rapid CD4+ T cell depletion. We have investigated the significance of EvT3 V3 sequences in determining the biological properties by construction of V3 recombinant viruses between EvT3 and SIVmac239. Our data have documented that EvT3 V3 sequences are responsible for the inability to replicate in human T cell lines in vitro, and they are also essential for induction of CD4+ T cell depletion in vivo. Interestingly, EvT3 V3 is more positively charged and hydrophilic than SIVmac239 V3, which has a property similar to V3 of T cell-tropic HIV-1. Recently, it has been shown that T cell-tropic HIV-1 containing positively charged V3 sequences utilized CXCR4 as a co-receptor for virus entry. Consistent with the properties of V3 sequences, we found that EvT3 efficiently utilized CXCR4 for virus entry. In contrast, SIVmac239 utilized CCR5 but not CXCR4 similar to Mm-tropic HIV-1. Our data using V3 recombinant viruses indicated that the distinct co-receptor usages between EvT3 and SIVmac239 are also determined by their V3 sequences. These data indicate that SIV V3 sequences play important roles in determining the viral cell specificity, pathogenic potential and co-receptor usages. We are currently making efforts to identify the genetic determinant(s) in V3 determining the biological properties of SIV, and further investigating the biochemical basis underlying the interaction between V3 and co-receptor which is important in determining the cell specificity in vitro and possibly pathogenic potential in vivo.